Protein synthesis, or translation, directly couples genotype to phenotype in the cell. Translation occurs on the ribosome, an RNA-protein complex conserved in all forms of life. The ribosome is composed of two subunits that must work together as an intact complex in order to function. A complete understanding of protein synthesis will require an atomic-resolution movie of the intact ribosome during one cycle of amino acid addition to a growing polypeptide chain. In this regard, the ribosome is no different from other molecular machines, such as RNA and DNA polymerases, helicases, myosins and kinesins. All of these molecular machines use conformational changes as a basis for function. And in each case, many x-ray crystal structures at atomic resolution have been necessary to obtain clear mechanistic insights into function. Cryo-EM reconstructions and x-ray crystal structures have identified many moving parts within the ribosome that remain to be probed at atomic resolution due to a lack of high-resolution structural information. One functional state of the ribosome that is critical to many steps of translation, the ratcheted state, remains poorly understood at the molecular level. The ratcheted state of the ribosome is central to the process of moving messenger RNA (mRNA) and transfer RNA (tRNA) by one codon after each peptide bond is made, a process called translocation. The ratcheted state also plays an important role in ribosome recycling, the step in which ribosomes that have completed synthesis of one protein are dissociated into the two ribosomal subunits to promote reinitiation of translation. The ratcheted state is also a key target of many antibiotics. In the past four years of this grant, we have successfully used the model organism Escherichia coli for x-ray crystallographic and biochemical studies of the intact ribosome, bridging structural data with functional insights at atomic resolution. We obtained two crystal forms of the intact ribosome that diffract to atomic resolution, which resulted in several groundbreaking structural studies of the ribosome. Our focus on the E. coli ribosome allows us to compare our results directly to decades of biochemical and genetic data obtained with the E. coli translational machinery. We have now obtained atomic-resolution diffraction from crystals of the E. coli 70S ribosome in the ratcheted state. This proposal describes three strategies for probing both the structure and function of the intact E. coli ribosome based on this new breakthrough. 1. Determine the atomic-resolution structure of the intact 70S ribosome in the ratcheted state. 2. Determine atomic-resolution structures of the ratcheted ribosome in complexes with tRNAs, ribosome recycling factor (RRF), elongation factor G (EF-G), or antibiotics that target the ratcheted state. 3. Probe the effects of macromolecular crowding on the kinetics and thermodynamics of ribosome recycling. These experiments will build on the structural insights gained in the present funding period and in Specific Aims 1 and 2.